This project aims to understand biomembrane structure and function with the aid of a recent lab-achieved breakthrough in control of membrane phospholipid and sphingolipid composition via cyclodextrin-catalyzed lipid exchange. This permits preparation of lipid vesicles mimicking natural membranes closely in terms of both lipid composition and, for the first time, lipid asymmetry, the difference in the lipid composition in the inner and outer lipid layers characteristic of many natural membranes. The method is also being extended to control of lipid composition in living cells. These methods are being applied to solve long-standing issues of membrane domain formation. We and collaborator Dr. Deborah Brown proposed in 1994 what remains the working model in the field: cell membrane domains form due to segregation of sphingolipid-cholesterol rich liquid ordered (Lo) domains from unsaturated lipid rich liquid disordered domains. Although such domains are readily observed in artificial lipid vesicles, and careful studies document their presence in living cells, domains remain controversial and poorly characterized. This project will use lipid exchange to overcome roadblocks to progress in studies of membrane domains. First, studies using asymmetric lipid vesicles that mimic cell membranes much more closely than the symmetric vesicles employed in the past will define the rules governing domain properties and formation by lipids and proteins. This includes testing the long-standing hypothesis that lipid-induced signal transduction across membranes can result from coupling between physical properties of lipids in the inner and outer lipid layers of a membrane. Knowledge gained from these studies will reveal how to manipulate lipids and proteins in cells to control domain formation and protein association with domains, and thus how to explore their function. Second, domains will be studied with more tractable living systems and methods. We found that the cholesterol-containing bacterium Borrelia burgdorferi, the causative agent of Lyme disease, has domain size sufficient for visualization by electron microscopy and facile fluorescence domain detection using FRET, plus accessibility to altering sterol chemical structure to allow controlled modulation of domain formation. This made it possible to unequivocally identify bacterial Lo domains in vitro and in vivo. Studies will be extended to other pathogenic bacteria likely to contain Lo domains: Helicobacter pylori, the cause of ulcers and some cancers, and S. aureus, drug-resistant strains of which (MRSA) are a major public health threat. Studies will define the properties of bacterial domains, the principles behind their formation, and potential biomedical implications. Studies will then be extended to mammalian cells taking advantage of our discovery of a cyclodextrin that can fully exchange plasma membranes outer leaflet lipids with exogenous phospholipid and sphingolipid without disturbing cell sterols. Using this, how altering lipids modulate domain formation, properties, and function will be defined. This method will also be used to investigate plasma membrane lipid asymmetry and its function. Further development of the method will broadly impact biomembrane studies.